In chromosomal DNA of higher eukaryotic organisms, the 5-position of cytosine (C) among the bases constituting DNA may be methylated in some cases. Methylation of DNA in the higher eukaryotic organisms functions as a mechanism for controlling the expression of genetic information. For example, when a region with many CpG sequences which is generally found in promoter regions of genes (CpG island) is methylated, transcription of these genes is suppressed. On the other hand, when the CpG island is not methylated, transcription factors can bind to the promoter region and then the genes can be transcribed.
Thus, methylation of DNA is one of the regulating mechanisms of the gene expression. Due to this, methylation of DNA plays important rolls in various physiological and pathological phenomena such as early embryogenesis, tissue specific gene expression, genomic imprinting and inactivation of X chromosomes which are characteristic phenomena of mammals, stabilization of chromosomes, the timing of DNA replication and the like. Recently, it has been found that methylation of DNA is strongly relevant to cancers and other diseases.
A methylation specific polymerase chain reaction method is known as the method for analyzing methylation of DNA (see James G. HERMAN et al., Methylation-specific PCR:A novel PCR assay for methylation status of CpG islands, Proc. Natl. Acad. Sci. USA, Vol.93, pp. 9821-9826, September 1996). In this method, a methylated cytosine and non-methylated cytosine are differentiated by converting the non-methylated cytosine to another base with a reagent that can convert bases such as bisulfites. However, it is known that such conversion treatment of DNA with the base-converting reagent degrades most of DNAs in biological samples due to the chemical reaction.
Recently, a method for detecting methylation of cytosine in a DNA sample is disclosed in which the DNA sample is hybridized with a guide probe such that the cytosine suspected of being methylated in the DNA sample forms a bulge structure or a mismatch, the methylated cytosine in the bulge structure or the mismatch is then specifically oxidized with an oxidizing agent such as osmates, and the oxidized product is detected (see WO 2006/132022). This method allows the detection of a methylated cytosine by utilizing the fact that the methylated carbon atom in pyrimidine ring on the methylated cytosine is liable to be oxidized, thereby detecting the oxidized product.
When a DNA sample is treated with an oxidizing agent, methylated cytosines other than the target methylated cytosine and thymines that have a methyl group similar to methylated cytosines are also oxidized. In order to avoid the oxidization of bases other than target bases, base pairs are formed between the guide probe and the bases in the DNA sample other than the target bases by hybridization of the guide probe and the DNA sample.
However, when the bulge structure or the mismatch is formed between the DNA sample and the guide probe, a steric distortion may be generated in the double helix structure after hybridization. More specifically, in the bulge structure, the cytosine which is targeted for the detection of methylation projects from a double-stranded DNA formed by hybridization of the DNA sample and the guide probe. Due to this steric reason, the base adjacent to the bulge structure in the DNA sample is difficult to form a base pair with the corresponding base in the guide probe. In case of the guide probe which forms the mismatch, the base adjacent to the mismatch is also difficult to form a base pair with the corresponding base in the guide probe because of the steric hindrance of the base which does not form a base pair with the target cytosine for detection. When thymine is present in a region adjacent to the cytosine suspected of being methylated and when the DNA sample contains CpG island, in particular, it is highly probable that the steric distortion is generated. Accordingly, the bases other than the target base in the DNA sample and the guide probe may not sufficiently form base pairs, and thymines which are not targeted may possibly be oxidized.
When the detection of methylated cytosine is used as a diagnosis of cancers or other diseases, highly precise detection is required in view of the reliability of the diagnosis results. Therefore, there has been a need to make an improvement in accuracy of the detection of methylated cytosines.